Abstract: Artemisinin combination therapies are a critical component to malaria control. The emergence of artemisinin resistance in Africa would spell a global public health disaster, potentially reversing gains made in control efforts. Unfortunately, several groups are now reporting small increases in parasite clearance times, the clinical marker of resistance, at several sites in Africa. Recently, multiple mutations in the K13 propeller region of PF3D7_1343700 have been associated with artemisinin resistance in Asia. Importantly, these mutations were shown to have arisen multiple times independently, raising concern for de novo resistance mutations arising in Africa. This discovery pushed us to look for mutations in the K13 gene among African parasite isolates. Using a pooled sequencing approach, we identified an array of novel mutations in the K13 propeller, including some at startlingly high frequency, but few shared with Asia. This raised critical questions concerning the impact of the African mutations in K13 and the development of artemisinin resistance. In order to better understand the significance of these findings, here we propose an approach to study the selection of African K13 alleles by ACTs and to phenotype these mutations by leveraging previously collected samples and new in vitro tools, without having to conduct large and expensive in vivo efficacy studies. Using previously collected samples from the 2007 and 2013 Demographic Health Surveys (DHS), we will hybrid selection and whole genome sequencing of pooled isolates to evaluate for changes in frequency of mutations in the DRC as ACT use has increased and became common, with >60% of cases being treated with ACT over the last 4 years. Increasing frequency of mutations, while controlling for parasite demographics and natural genetic variation, would raise concerns that mutations are under selection. However, direct evidence would be required to determine if these mutations alter susceptibility to artemisinin. In order to provide this, we will use reverse genetics to assess the impact of candidate mutations (those already at high frequency in our previous work and those suspicious for resistance in other studies) or mutations that increase in frequency between 2007 and 2013 in this study in vitro by transfecting mutations into parasite lines and using the recently developed Ring Survival Assay (RSA). This assay has been correlated to the in vivo phenotype. This proposal is innovative because it will be the first time we use a molecular marker of antimalarial resistance early during the emergence of resistance, and due to the use of several novel approaches, including our hybrid capture of malaria DNA from blood spots. It also leverages current advances in reverse genetic engineering of the parasite. This project is significant as it will provide critical insight into the current situation concerning artemisinin resistance in Central Africa and an increased understanding on what described polymorphism mean in context of resistance. These findings will have important impacts for both our understanding of parasite biology, as well as for control and elimination strategies.